Enzyme conjugates and method of preparation and use

ABSTRACT

Novel enzyme conjugates useful in immunoassay methods are prepared with the use of a novel coupling reagent of N-succinimidyl (4-iodoacetyl) aminobenzoate by reacting the coupling reagent, firstly, with an amino-containing macromolecule, and, thereafter, with a sulfhydryl-containing enzyme, the enzyme conjugate prepared in a high yield and of high specificity.

BACKGROUND OF THE INVENTION

Enzyme-macromolecule conjugates are typically used for the detection anddetermination of substances present in very low quantities; for example,nanogram quantities of substances in biological fluids, such as urineand serum. A wide variety of enzymes may be used to form the conjugate,but the enzymes selected are often those enzymes which can be detectedwith great sensitivity. The macromolecular portion of the conjugatepresents a wide variety of amino-containing compounds, including, butnot limited to, nucleic acids, proteins, hormones, antigens andallergens, which are characterized in containing amino groups.

Enzyme conjugates are prepared in a conjugation reaction with apolyfunctional coupling reagent which links the enzyme and macromoleculetogether by reaction with one or more of the reactive groups in thereactants. In the preparation of enzyme conjugates, it is most desirableto produce enzyme conjugates of high stability, high specificity andgood reproducibility.

In some coupling reactions to prepare enzyme conjugates, it has beensuggested to employ a conditioner compound, such as a polyamine, toimprove specificity of the enzyme conjugate, with resulting improvementin the detection method due to low signal-to-noise ratio duringdetection in the immunochemical test. The preparation of enzymeconjugates, employing prior-art coupling reagents with conditioners, isdescribed in U.S. Pat. No. 4,002,532, issued Jan. 1, 1977, herebyincorporated by reference.

A new coupling agent has been described for the preparation of anenzyme-coupled insulin conjugate for use in the immunoassay of insulin.The coupling agent is meta-maleimidobenzoyl N-hydroxysuccinimide ester(MBS), a bifunctional reagent which acylates the amino groups of theinsulin by reaction with the N-hydroxysuccinimide ester group and byforming thioester bonds with the enzyme by addition of the thiol groupsto the maleimide group. This coupling agent has been employed inpreparing an enzymatically active and immuno-reactiveB-D-galactosidase-MBS-insulin conjugate (see J. Biochem., 79, 233-236(1976), "Enzyme Coupled Immunoassay of Insulin Using a Novel CouplingReagent", Kitagawa, T and Aikawa, T, hereby incorporated by reference).Although the MBS coupling reagent is satisfactory in some respects, itis desirable to obtain enzyme conjugates of greater stability andgreater specificity and sensitivity.

SUMMARY OF THE INVENTION

My invention relates to novel coupling reagents and their method ofpreparation, which reagents are useful in the preparation of stableenzyme conjugates, to the method of preparing the enzyme conjugatesemploying my coupling reagents, and to the use of such enzyme conjugatesin immunoassay methods.

My polyfunctional class of coupling reagents, to form enzyme conjugateswith a high yield and specific activity, comprises, in the genericsense, a reagent which reacts, firstly, with the amino groups of themacromolecule and then with the sulfhydryl groups in the enzyme, and arerepresented by the formula: ##STR1## where X is a halogen, preferablyiodine; n is a whole number of from about 1 to 8, preferably 1 to 4; andR represents any radical which is capable of reacting with the aminogroups of the macromolecule, such radical including, but not beinglimited to: ##STR2## wherein R₁ is hydrogen or an alkyl group, such asmethyl.

Other amino-reacting groups may be employed as the R radical.

The most preferred coupling reagent is N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) having the structural formula: ##STR3## The SIABreagent produces enzyme conjugates with unexpectedly high yields andspecific immunochemo, enzyme and conjugate activity. Enzyme conjugatesprepared with SIAB, in comparison to other prior-art coupling reagents,such as MBS, provide enzyme conjugates of high stability and highspecific conjugate yields.

Specific useful coupling reagents include, but are not limited to:iodoacetic acid; iodoacetic anhydride; and N-hydroxy-succinimide esterof iodoacetic acid.

Iodoacetic acid reacts with --NH₂ groups in the presence of awater-soluble carbodiimide, such as 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide. Iodoacetic anhydride reacts directly with --NH₂ groups. Inthe other cases, my coupling reagents are prepared by reacting theselected halo organic acid or acid anhydride in a solvent with the Ramino-reacting portion of the molecule, and the reagent is thenrecovered. For example, SIAB is synthesized by reaction of iodoaceticanhydride with paraaminobenzoic acid in an organic solvent, such asdioxane. The resultant intermediate compound is crystallized and theactive ester is formed between the intermediate andN-hydroxysuccinimide, with a carbodiimide in an organic solvent; forexample, with dicyclohexylcarbodiimide in tetrahydrofuran. The resultantproduct, which may be crystallized, is SIAB.

The enzyme conjugates of my invention are prepared by a conjugationreaction between the amino-reacting substituent of my coupling reagentand the macromolecule, and, thereafter, reacting the halo-reacting agentwith the sulfhydryl groups of the enzyme. It is essential in thepreparation of my enzyme conjugates that the reaction sequence be,firstly, the reaction of the R amino-reacting group, such as thesuccinimidyl group, with the amino group of the protein or othermacromolecule, and, thereafter, reaction of X with sulfhydryl groups ofthe enzyme; otherwise, the R group would react with the amino group ofthe enzyme, resulting in lower yields and a lack of specificity in theresulting enzyme conjugate product so prepared.

For example, my coupling reagent provides for the preparation of anenzyme conjugate with a high degree of specific coupling, as representedby the following illustrative reaction with SIAB: ##STR4##

Generically the method of preparation would be: ##STR5##

Typically SIAB is reacted in an aqueous solution with amino groups ofthe macromolecules under mild conditions. For example, SIAB is reactedwith immunoglobulin in 0.05 M phosphate buffer, pH 7.0. The resultantimmunoglobulin derivative is then reacted with an enzyme which containssulfhydryl groups. One such enzyme is Beta D galactosidase (BG) isolatedfrom E. coli. BG is reacted with the derivatized immunoglobulin undermild aqueous conditions. The resulting BG macromolecule conjugates soprepared with my coupling reagent have a high degree of enzyme activity,immunological specificity, and are obtained in extremely high yields;for example, virtually all the immunoglobulin or other macromolecule andBG are stoichiometrically conjugated to each other. The yield of BGconjugate so achieved is surprisingly high. The conjugates of BG witheither antigens or antibodies produced with SIAB are useful in enzymeimmunoassays for various antigens or antibodies, including hepatitis,serum proteins, hormones and drugs.

Any SH-reactive enzyme may be used in my method, such as, for example,acid and alkaline phosphatases, alcohol dehydrogenase catalase, glucoseand galactose oxidases, a- and B-galactosidases, lactate dehydrogenase,lysozyme, luciferase, peroxidases, ribonuclease, rodhanase andesterases.

Useful NH₂ -reactive macromolecules include deoxy and ribonucleic acids,viral proteins, allergens, immunoglobulins, blood group substances,transplantation antigens, carcino embryonic antigen, alpha-fetoproteinand other tumor specific antigens, growth hormone and other polypeptidehormones.

The SIAB is particularly useful in the preparation ofimmunoglobulin-bacterial enzyme B-D-galactosidase conjugates. The enzymeconjugates of the invention are used for the detection of antigens,antibodies, allergens, hormones and other macromolecules in lownanogram, or less, quantities by immunoassay techniques.

My unique enzyme conjugates find particular use in those testexperiments and detection techniques relating to macromolecules,particularly to the detection and determination of nanogram quantitiesof substances in biological fluids, such as the detection of antibodies,antigens, allergens, hormones and the like. In particular my SIAB enzymeconjugates fine use in enzyme immunoassay techniques for hepatitis. Inprior-art techniques, repeated washings of the patient's serum on thesolid support; that is, an antigen disc, are required to removenonspecific, absorbed serum proteins. However, the use of SIAB conjugateenzymes makes feasible a one-step enzyme immunoassay method. Limitedwashing or no washing is possible with my SIAB enzyme conjugates, sinceall or substantially all of the specific-form enzyme is bound to theantibody.

My enzyme conjugates may be employed in the prior-art techniques for thedetection of antibodies; for example, as set forth in U.S. Pat. No.4,002,532 (supra) wherein the detection method comprises adhering, suchas by absorption, to a solid support or absorbent material, such as adisc, which binds or absorbs the antibodies or other macromolecules tobe detected; adding to the solid support material a fluid, such as serumor urine, to bind the antibodies therein to the support material used;for example, incubating the serum with the disc; washing the supportmaterial to remove unbound components of the fluid, such asnonspecifically bound and absorbed serum proteins, from the discsurface; adding the enzyme conjugate to the washed support, such as byincubation of the disc with an enzyme-active SIAB protein conjugate, sothat the specific enzyme conjugate is bound to the support in proportionto the quantity of disc-bound antibodies, and thus is a measure of thepatient's IgE or other macromolecules to be quantitated; optionallywashing the support to remove unbound unspecific enzyme conjugate fromthe support disc; and determining the enzymatic activity of the boundenzyme conjugate as a measure of the amount of bound antibodies.

In such a detection method, the high specific yield of my enzymeconjugate provides for a high degree of coupling with the supportmaterial in comparison to prior-art techniques, as indicated, forexample, in the unexpectedly high signal-to-noise ratio in the detectionmethod; that is, the enzyme conjugates of my invention increasesensitivity and specificity by specific bonding of the enzyme conjugateto the solid support, and permit increased sensitivity in the detectionof antibodies with lower quantities of enzyme conjugates and in thepresence of interfering substances not possible in the prior-art enzymeconjugates. For example, when employing the SIAB coupling reagent with ahuman serum containing a known amount of antigen, the enzyme conjugateis quite stable, and a determination of the amount of bound enzyme isabout three and one-half times more than with the MBS coupling reagentat about one-half the amount of enzyme to provide a specific conjugatewith an improvement of about 15 fold.

My invention will be illustrated by specific and preferred examples;however, it is recognized that various changes and modifications can bemade in such examples without departing from the spirit and scope of myinvention.

DESCRIPTION OF SPECIFIC EMBODIMENTS EXAMPLE 1

Preparation of SIAB Coupling Reagent

N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), the preferred couplingreagent of my invention, was prepared by the following reaction:##STR6##

354 mg of cpd I (10³ μmoles) were dissolved in 5 ml dioxane and added to68.6 mg of cpd II (500 μmoles) in 2.5 ml of dioxane and were reacted for5 hours at room temperature (20°-25° C.) in the dark and then at 4° C.for 2 days. A white flocculent precipitate (cpd III) was isolated bycentrifugation and triturated with ether (0.5 ml) three times. Theresulting white powder was dried with hot air with a yield of 160 mg.Cpd IV (86.2 mg; 4×10⁻⁴ moles) was added to a solution of cpd III (128mg; 4×10⁻⁴ moles) and cpd V (48.5 mg; 4×10⁻⁴ moles) and reacted intetrahydrofuran THF (3.35 ml) and placed at 4° C. for 20 hours. Aprecipitate was removed and the supernatant liquid was recovered andevaporated to dryness and triturated with ether, and pale yellowcrystals were recovered of impure SIAB (cpd VI), yield 135 mg (79.5%yield), mp 172°-175° C. The impure cpd VI was recrystallized from methylalcohol and was washed twice with diethyl ether to provide whitecrystals of SIAB having an mp of 194° to 196° C. (decomp). Confirmationof this SIAB composition was made by elemental analysis.

EXAMPLE 2

Preparation of SIAB Enzyme Conjugates

Rabbit antibodies against sheep immunoglobulin (RaShIg) were conjugatedto beta-D-galactosidase (BG) with SIAB to provide an enzyme antibodyconjugate. The purified RaShIg and the BG were obtained as set forth inExample 1, U.S. Pat. No. 4,002,532. A mixture of RaShIg (3.9 mg,2.63×10⁻⁸ moles) and SIAB (30 of 3.6 mg/ml of THF) was prepared (SIABwas added to RaShIg) in an aqueous 0.05 M sodium-phosphate-bufferedsaline solution (pH 7.0) and the mixture was permitted to react at roomtemperature overnight; that is, 12 hours in the dark, to provide anacylated RaShIg product, with some of the amino groups of the antibodiesreacted with the active succinimidyl group of the SIAB coupling reagent.The reaction was quenched by the addition of glycine (30 , 4.5×10⁻²moles) to the acylated RaShIg for 3 hours at room temperatures in thedark. To the quenched RaShIg was added BG (25×molar excess of RaShIgover BG), with the solution adjusted to a pH of 7.8 at 4° C. for 2 days.Thereafter, the reaction of the iodine radical with the sulfhydryl ofthe enzyme BG was quenched by the addition of 2-mercaptoethanol (4×10³M), and the mixture was maintained at room temperature for 3 hours. TheRaShIg-BG conjugate so obtained was diluted, clarified and recovered.There was not detectable loss in activity of enzyme as a result of thecoupling procedure.

EXAMPLE 3

Preparation of Iodoacetyl N-Succinimide (INS) ##STR7##

EXAMPLE 4

Comparison of RaShIg-BG Conjugates

The conjugate so prepared was prepared in a buffered enzyme solution ofstandard units (10,000) of enzyme activity per ml and tested, as in U.S.Pat. No. 4,002,532 (column 5, line 55-column 6, line 1), to determinethat the conjugate exhibited a high degree of specificity as illustratedby the S/N ratio shown in the table below, when compared with the sameconjugates prepared in a similar manner, but with prior-art couplingagents MBS and W-R agent (see U.S. Pat. No. 4,002,532, Example 1).

                  TABLE I                                                         ______________________________________                                        Comparison of S/N of Sheep Immunoglobulin (ShIg)                              Conjugates of Beta Galactosidase (BG)                                                      Coupling                                                         Units of ShIg-BG                                                                           Reagent                                                          Added Per Disc                                                                             SIAB*     MBS*       W-R                                         ______________________________________                                        1            94 ± 7 41 ± 1  67 ± 4                                   10           63 ± 2 58 ± 1  52 ± 2                                   100          32 ± 2 18 ± 0  20 ±  1                                  ______________________________________                                         *Numbers are S/N ± standard deviation of duplicate measurements. S is      binding to immuno specific RaShIg discs. N is binding to normal               nonspecific Rabbit RIg discs.                                            

The maximum S/N ratio for SIAB is almost twice the S/N for the MBScoupling agent, and the S/N for SIAB was rising monotonically while theMBS S/N went through a maximum value. Thus, SIAB conjugates could beused at even lower than unit concentrations to provide even better S/Nratios. The S/N ratios establish that the SIAB conjugates are highlyspecific in comparison to prior-art conjugates. This is borne out by acomparison of SIAB and W-R conjugates in a test for HB_(s) Ag (TableII).

                  TABLE II                                                        ______________________________________                                        Comparison of Conjugates* of Beta Galactosidase (BG) in a                     Test for Hepatitis B-Surface Angiten (HB.sub.s Ag)                            HB.sub.s Ag (ng)                                                                         SIAB**     W-R**      SIAB/W-R                                     ______________________________________                                        0          0          0          --                                           0          0          0          --                                           3.13       .00462     .00115     4.02                                         3.13       .00322     .00196     1.64                                         6.25       .00815     .00226     3.61                                         6.25       .00645     .00156     4.13                                         12.50      .01435     .00786     1.83                                         12.50      .01275     .00766     1.66                                         25.00      .02035     .01376     1.55                                         25.00      .02145     .01396     1.54                                         ______________________________________                                         *Conjugates are Rabbit antigoat Ig coupled to beta galactosidase used to      detect goat antiHB.sub.s Ag.                                                  **Numbers are units of BG bound to each immunosorbent disc.              

The average SIAB/W-R is 2.5; that is, an average of 2.5 times more SIABconjugate was specifically bound. The procedure followed was that setforth in the Weltman et al patent (supra).

What I claim is:
 1. A method of preparing an enzyme conjugate, whichmethod comprises reacting at least one of the amino groups of anamino-containing nonenzyme macromolecule with an amino-reactive radicalR of a coupling reagent having the formula: ##STR8## wherein X is ahalogen; n is a whole number of from 1 to 8; and R is an amino-reactingradical to form a reagent-macromolecule compound, and, thereafter,reacting the sulfhydryl-reacting halogen of the couplingreagent-macromolecule; with the sulfhydryl groups of an enzyme to forman enzyme-conjugate compound of the enzymatically active enzyme and theimmuno-active macromolecule conjugately linked by the coupling reagent.2. The method of claim 1 wherein the amino-reactive radical is selectedfrom the group consisting of: ##STR9## wherein R₁ is hydrogen or analkyl group.
 3. The method of claim 1 wherein the coupling reagentcomprises a haloacetyl N-succinimidyl compound.
 4. The method of claim 1wherein the coupling reagent comprisesN-succinimidyl(4-iodoacetyl)aminobenzoate.
 5. The method of claim 1wherein the amino-containing macromolecule comprises a nonenzymeproteinaceous macromolecule selected from the group consisting ofantibodies, antigens, allergens, hormones, immunoglobulin and serumsubstances.
 6. The method of claim 1 wherein the enzyme comprisesbeta-D-galactosidase.
 7. The method of claim 4 wherein the enzymecomprises B-D-galactosidase and the macromolecule comprisesimmunoglobulin.
 8. The enzyme conjugate produced by the method of claim4.
 9. The B-D-galactosidase-immunoglobulin conjugate produced by themethod of claim
 6. 10. The enzyme conjugate produced by the method ofclaim
 1. 11. The method of claim 4 wherein the X radical is iodine. 12.The method of claim 4 wherein the coupling reagent comprises iodoacetylN-succinimide.
 13. The method of claim 4 which includes quenching thereaction of the macromolecule with glycine and, thereafter, quenchingthe reaction of the enzyme with 2-mercaptoethanol.
 14. A method ofpreparing an enzyme conjugate of a high degree of enzyme activity,immunological specificity and at high yields, which method comprisesreacting at least one of the amino groups of an amino-containingnonenzyme macromolecule with N-succinimidyl(4-iodoacetyl)aminobenzoateas a coupling reagent to form a reagent-macromolecule compound and,thereafter, reacting a sulfhydryl-containing enzyme with the couplingreagent-macrolecule to form an enzyme-conjugate compound ofenzymatically active enzyme and an immuno-active macromoleculeconjugately linked by the coupling reagent.
 15. The method of claim 14wherein the macromolecule comprises immunoglobulin and the enzymecomprises beta-D-galactosidase.
 16. The method of claim 14 whichincludes quenching the reaction of the macromolecule with glycine and,thereafter, quenching the reaction of the enzyme with 2-mercaptoethanol.17. The conjugate enzyme produced by the method of claim 15.